Introduction

In recent years there has been increasing appreciation of the complex nature of post-transcriptional control of eukaryotic gene expression through regulation of pre-mRNA and mRNA processing (1,2). Throughout the life of a given transcript, it can be influenced by numerous RNA binding proteins (RBPs) that associate with transcripts in the nucleus and/or cytoplasm. In many cases these RBPs bind in a sequence-specific manner to RNA sequence elements often referred to as regulatory cis-elements. Such cis-elements can be found in 5′ and 3′ untranslated regions (UTRs), coding exons, and introns, and identification of the RBPs that bind these cis-elements is a frequent goal. Complicating the identification of functionally relevant RNA-protein interactions is the fact that, although many such interactions are highly sequence-specific, many abundant cellular RBPs (i.e., hnRNP proteins and SR proteins) have degenerate binding sites and/or bind promiscuously to pre-mRNA or mRNA transcripts (3). In addition, regulatory cis-elements are frequently bound by a complex of proteins that each recognize distinct sequence motifs or that may associate indirectly with RNA.

The identification of regulatory RBPs is commonly carried out through the use of RNA affinity chromatography by immobilizing RNA cis-element on a solid matrix. Following in vitro incubation with cellular extracts, the proteins that bind to the RNA can then be identified by mass spectrometry. A convenient and popular method for covalently coupling RNA to agarose beads is carried out by oxidation of the 3′ end of the RNA with sodium periodate followed by direct coupling to adipic acid dihydrazide agarose beads (4,5,6,7). We recently used this approach to identify hnRNP M as one RBP that associates with an intronic RNA cis-element that plays a role in regulation of fibroblast growth factor receptor 2 (FGFR2) splicing (8). The majority of proteins identified by these approaches are abundant RBPs, such as the core hnRNP proteins (5,9,10,11,12,13,14). The high abundance of these proteins, together with their presence in common RNP particles, raises the possibility that their identification in RNA affinity columns may result not only from high-affinity sequence-specific interactions, but also by lower-affinity, non–sequence-specific binding and through indirect interactions as well. For example, immunoprecipitation with a specific hnRNP antibody (i.e., against hnRNPA1) can be used to co-purify most of the other core hnRNP proteins in cellular hnRNP (15). Thus, even where one protein may bind specifically to an immobilized RNA, proteins with which it interacts may also indirectly be bound to the same column. Such indirect interactions might consist of protein-protein interactions, but could also occur if the proteins are both bound to common RNAs. Such RNA dependent protein-protein “tethering” interactions have been previously described and, as such, inclusion of an RNase treatment step is often recommended for biochemical investigations of binding partners for RNA binding proteins (16,17,18). An additional consideration is that extracts used in these purifications contain cellular RNase activity that may mask identification of some RBPs due to partial degradation of some sequences within the RNA column.

We demonstrate a procedure for the use of RNA affinity chromatography using RNase-resistant RNAs. Among the 2′ pentose ring substitutions that have been shown to render RNA resistant to specific RNases, 2′ fluorinated RNA can be synthesized using standard in vitro synthesis protocols using T7, T3, or SP6 RNA polymerases through the use of 2′-fluorine-CTP (2′-F-dCTP) and 2′-fluorine-UTP (2′-F-dUTP) in place of CTP and UTP (19). We show here that this approach facilitated the use of RNase-treated extracts for affinity purification without significant degradation of the immobilized RNA. The fluorine-substituted RNAs could be covalently linked to adipic acid dihydrazide beads using the same protocol we have previously used for unmodified RNA. Using an RNA that was previously shown to be directly bound by hnRNP M, we show that the same RNA is also recognized by this RBP when it has been fluorine modified.

Materials and methods

Plasmid construction

The constructs used as templates during in vitro transcription, pDP19RC-ΔEE−ISE/ISS-3-WT and pDP19RC-ΔEE-ISE/ISS-3-BS, were made using standard cloning techniques as described previously (8).